Urinary protein profiling for biomarker discovery in diabetic nephropathy

Renal disease in patients with Type II diabetes is the leading cause of terminal renal failure and a major healthcare problem. Diabetic renal damage might be reflected by a change in urinary proteins and polypeptides excretion at a very early stage. The characterization of urinary polypeptides is of great clinical interest and significance, yielding to a better understanding of the changes within the kidney during the development of diabetic nephropathy (DN).Proteomics has now become an emerging field in the post-genomic area and offers the opportunity of large-scale protein analysis. Two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) combined with mass spectrometry (MS) is commonly used for protein separation and to determine the molecular weight of polypeptides and proteins. Surface-Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS), which represents the succesful combination of retentate chromatography and MS, is an alternative technology employed for high-throughput analysis of biological samples.The aim of this study was to identify urinary protein patterns in Type II diabetic subjects, that may serve as diagnostic tool for the early recognition and development of DN.In our study, we included 25 Type II diabetes mellitus patients with no nephropathy, 25 Type II diabetes mellitus patients with nephropathy (identified by serum creatinine level < 2mg/dl and microalbuminuria) and 20 healthy volunteers. Morning midstream urine samples were collected, centrifuged to remove cell debris and precipitated with acetone/methanol at -20°C. After centrifugation, the protein pellet was resuspended in a specific solubilization buffer and finally 100 micrograms of protein were subjected to 2DE-PAGE. Silver-stained gels were analyzed by the PDQuest® Image Analysis software, differential proteins were cut and identified by LC-ESI-MS/MS.For protein profiling with SELDI-TOF-MS, urine samples were analyzed using immobilized metal affinity capture (IMAC30), strong anionic exchanger (Q10), weak cation exchanger (CM10), reverse phase (H50) ProteinChip® Arrays and sinapinic acid (SPA) as matrix, according to the manufacturer’s instructions (Ciphergen, USA). Urinary protein profiles were collected in the range 0-200.000 m/z, using different acquisition protocols and different laser energies. Statistical analysis was performed in the obtained spectra (Ciphergen Express 3.0) in order to identify peaks that showed significant differences among groups.Two characteristics proteins (prostaglandin-H2 D-isomerase and antithrombin-III) were identified in the urine of Type II diabetes patients compared to healthy individuals using 2DE-PAGE coupled to MS. In addition to these two proteins, the following proteins were identified in urine of patients with Type II diabetes mellitus with nephropathy: vitamin-D binding protein, gelsolin, apolipoprotein A-IV, plasma retinol binding protein and lipocalin-1, with an increased amount of albumin.In conclusion, comparative data analysis discovered distinct proteins characteristic for diabetic patients and leads to the establishment of possible individual risk factors for the development of DN, which may contribute to clinical staging in the future. These techniques may also enable the identification of novel proteins related to the pathophysiology of DN, which may serve as therapeutical targets